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The global health field is significantly affected by viral infections, and sero-diagnosis is crucial in diagnostic virology. Various laboratory techniques such as nucleic acid detection, viral culture, and antigen detection are essential for diagnosing viral infections. Advances in science have led to the development of new immunologic and molecular techniques, enabling rapid and simplified diagnosis of different viruses. Timely and accurate identification of viral infections is vital for effective outbreak management. Immunological techniques, detecting viral antigens or antibodies, are widely used in diagnostic and epidemiological research, aiding in epidemic identification, appropriate diagnostic tests, vaccination programs, and detecting common and emerging viruses. However, traditional viral identification methods demand extensive technical expertise, time, and financial resources. Consequently, scientists worldwide are dedicated to developing precise diagnostic methods for viral diseases. Various innovative approaches are being explored, aiming to create more accessible, time-efficient, and cost-effective viral disease diagnosis methods, thereby benefiting low-income countries.
Indian Council of Medical Research, Regional Medical Research Centre for NE Region, Dibrugarh, Assam, India
Aniruddha Jakharia
Indian Council of Medical Research, Regional Medical Research Centre for NE Region, Dibrugarh, Assam, India
Pratibha Singh
Indian Council of Medical Research, Regional Medical Research Centre for NE Region, Dibrugarh, Assam, India
Siraj Ahmed Khan
Indian Council of Medical Research, Regional Medical Research Centre for NE Region, Dibrugarh, Assam, India
*Address all correspondence to: biswaborkakoty@gmail.com
1. Introduction
The Baltimore classification system and the International Committee on Taxonomy of Virus system both categorized viruses primarily on a small number of morphological characteristics. The Baltimore categorization system is based on two factors: the kind of genome (such as RNA or DNA, whether it is double- or single-stranded, etc.) and the mechanism of replication. Viral infection impacts millions of people every year and contributes to illness and death from serious, chronic, recurrent, or life-threatening disorders. The development of complicated and accurate mechanisms for detection of nucleic acid of virus and the antiviral antibody has potential to increase accuracy at various stages of the disease. Advancing diagnostic procedures in the past decades has significantly reduced the time and improved the accuracy of viral diagnosis, helping with patient care, disease control, and favorable impact on disease outcomes. Detection of viral antigen and antibody and viral nucleic acids are some of the more common methods for determining the presence of pathogens in humans. Most recently, the Real-Time PCR (qPCR) method has been widely employed in viral diagnostics due to its highly specific DNA/RNA detection and high-throughput quantification. Antiviral susceptibility testing, viral genotyping, and on-site care have all been developed as a result of rapid diagnosis leading to better and early treatment support. Laboratory-controlled serological tests have the benefits of high specificity, sensitivity, and differential diagnostic choices. Advancement in molecular and serological techniques has made it easier to diagnose viral infections [1, 2, 3, 4].
Every year, viral diseases claim the lives of millions of people worldwide, contributing to a significant global mortality rate. Due to seasonal influenza, there are billions of cases annually with 3–5 million cases of severe illness, leading to 290,000 to 650,000 respiratory deaths annually [5]. The swine influenza A (H1N1) in 2009 was declared a pandemic by WHO as it caused an estimated 284,400 deaths worldwide during the year. In 2010, rabies caused an estimated 61,000 deaths, with a substantial 84% of these occurring in rural areas. It is also estimated that 59,000 people die each year due to rabies [6]. During 2019 1.1 million people died due to viral hepatitis and it is also estimated that 354 million people live with hepatitis B or C worldwide. Acute hepatitis and chronic infection lead to liver cancer and cirrhosis and globally millions of people lose their live yearly [7]. More recently, the world faced unprecedented challenges in the form of the COVID-19 pandemic, triggered by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) [2]. World Health Organization (WHO) reported an alarming 771,407,825 confirmed cases of COVID-19 with 6,972,152 deaths till September 2023 (https://covid19.who.int/). In the face of these alarming statistics, the precise and early detection of viral infections emerges as a crucial aspect for effective disease management. Early detection not only enables prompt treatment but also plays a pivotal role in reducing treatment costs, lowering mortality rates, and gaining control over the spread of the disease. Furthermore, diagnostic methods such as variant identification and characterization facilitate early identification of viral variants, development of new anti-viral agents, and antimicrobial vaccines, which will augment the implementation of effective preventative measures. Conventionally, diagnostic methods relied on serological approaches, which revolutionized the diagnosis of viral infections in both humans and animals. However, these conventional methods have proven to be less sensitive, time-consuming, and difficult to perform in clinical samples other than blood. To overcome the limitations of conventional procedures, there is an urgent need for fresh and innovative approaches. Molecular methods, particularly those aimed at detecting viral nucleic acids, have emerged as promising solutions. These advanced techniques offer higher sensitivity and specificity, revolutionizing the diagnosis of viral infections. Embracing cutting-edge technology allows us to enhance the accuracy and efficiency of viral detection, ultimately leading to improved patient outcomes and effective control of viral outbreaks. Implementation of innovative approaches in viral diagnostics will serve as a significant step toward safeguarding the well-being of communities worldwide [5].
There have been many developments in diagnostic techniques (molecular and serological) for viral antigens or antibodies in the past many years. Diagnostic techniques have evolved through the ages and are still evolving (Table 1).
Restriction fragment length polymorphism analysis (RFLP)
2013
CRISPR system
1985
Immunoblotting assay
2015
VirCapSeq-VERT
Table 1.
Timeline for the development of various serological and molecular detection techniques constituting the mainstream approaches of both laboratory research and clinical diagnosis of viruses [8, 9].
2.2 Traditional method for virus identification
Isolating the virus through tissue culture, animal models, or embryonated chicken eggs was the traditional laboratory method for isolation and identification of medically important viruses [10].
2.2.1 Virus culture
Virus culture is the “gold standard” method for diagnosing culturable viruses. Viruses are cultivated in cell culture (Table 2) and cells infected with viruses exhibit morphological alterations that vary according to the virus’s properties (Figure 1). This technique is important, since it is the only approach that produces viable isolates for further characterization. However, it requires well-equipped bio-safety level laboratories and is time-consuming [10, 12, 13, 14].
Virus family
Virus
Cell line
Caliciviridae
Norovirus (NV)
HG23
Murine norovirus-1 (MNV-1)
RAW267.4
Coronaviridae
Human coronavirus 229E (229E)
MRC-5
SARS-CoV-2
Vero
Picornaviridae
Hepatitis A virus (HAV)
FRhK-4
Enterovirus 71 (EV71)
Vero
Human rhinovirus strains 18, 51, and 68 (HRV 18, 51, and 68)
HeLa
Adenoviridae
Adenovirus 1
Human embryo kidney cell
Hepadnaviridae
Hepatitis B virus
HepaRG
Table 2.
Viruses and respective culturable cell lines [11].
Figure 1.
(A). MDCK cell line culture (B). Infection with respiratory sample (C). Hemagglutination inhibition assay (D). Serial dilution method for virus culture.
2.2.2 Microscopy
Microscopy is considered a classic method for diagnosis/identification of viruses through direct visualization of viruses. Different types of microscopy are as follows:
2.2.2.1 Light microscopy
The light microscope, a vital tool in biology, identifies and magnifies objects by transmitting light through lenses. Replicating viruses induce distinctive histological changes in infected cells, often forming inclusion bodies, such as Negri bodies in rabies and cytomegalic inclusion bodies in CMV infections. These changes serve as diagnostic indicators in virology [15].
2.2.2.2 Electron microscope
Electron microscopy (EM) is a technique that allows direct visualization of viruses in clinical specimens and viral isolates. It is an essential tool in the detection, analysis of virus replication, and investigation of virus-host cell interactions. Identification by EM is based on morphological features of the virus, such as size, shape, and ultra-structural features. The constraint of electron microscopy is that sufficient viruses must be present (approx. 105–106 particles/mL). EM is not used widely for routine diagnosis because it is expensive and gives black-and-white images, and only live specimens can be visualized [6].
The natural immune response to viral infection has served as the foundation for the development of serological/immunological diagnostic methods. Antigen-antibody complex formation is the foundation for serological assays for the detection of whole viruses or viral antigens/viral antibodies, etc. Several assay procedures were developed for viral antigen or antibody detection:
2.3.1 Enzyme immunoassay
The term “enzyme immunoassay” is also referred to as the enzyme-linked immunosorbent assay (ELISA). It is more sensitive and capable of detecting ≤1 nanogram of viral antigen or antibodies per ml in the clinical specimen. The basic concept of this technique is to identify/detect the antigen or antibody of interest from the rest of the component present in the assay specimen/sample (Figure 2) [16].
Direct ELISA- In a direct ELISA, the antigen (sample/analyte) is immobilized on the surface of the multi-well plate and blocking is done with suitable blocking agents like BSA to avoid non-specific binding. Sample is added followed by addition of enzyme-conjugated-specific antibody which directly binds to the antigen of interest. Upon addition of substrate, there is a change of color which helps in the detection of antigen of interest in the sample. Proper washing steps are involved between each step except after the addition of substrate.
Indirect ELISA- An indirect ELISA is similar to direct ELISA, specific antigen is immobilized on the surface of the multi-well plate, and blocking is done similarly as in direct ELISA. The sample/analyte (primary antibody) is added, which binds to the immobilized antigen. The secondary antibody (anti-immunoglobulin antibody) conjugated with enzyme is added, which binds to the primary antibody. Similarly, direct ELISA substrate addition is done for detection.
Sandwich ELISA- It is the most commonly used ELISA test for diagnostic purposes. It requires two antibodies specific for different epitopes of the antigen. These two antibodies are normally referred to as matched antibody pairs. Capture antibody is immobilized on the surface of the multi-well plate, and blocking is done. The sample/analyte is added, which binds to the immobilized antibody. Followed by the addition of detection antibody conjugated with enzyme. Similarly, direct ELISA substrate addition is done for detection.
Competitive ELISA-Competitive ELISAs are commonly used for small molecules when the protein of interest is too small to efficiently sandwich with two antibodies. It is used to test for the presence of an antibody specific for antigens in the test serum. This type of ELISA utilizes two specific antibodies, an enzyme-conjugated antibody and another antibody present in the test serum (if the serum is positive). Combining the two antibodies into the wells will allow for competition for binding to antigens. The presence of a color change means that the test is negative because the enzyme-conjugated antibody bound the antigens (not the antibodies of the test serum). The absence of color indicates a positive test and the presence of antibodies in the test serum. The competitive ELISA has a low specificity and cannot be used in dilute samples. However, the benefits are as follows: It can measure a large range of antigens in a given sample, it can be used for small antigens, and it has low variability. Example—West Nile virus, Hepatitis A virus antibody, and Hepatitis B virus antibody.
2.3.2 Radioimmunoassay and time-resolved Fluoroimmunoassay
Enzyme immunoassay (EIA) precedes radioimmunoassay (RIA). The sole difference is that the label is a radioactive isotope like 125I rather than an enzyme and a gamma (γ) counter is used to quantify bound antibodies. A known quantity of antibodies is combined with radiolabeled antigens that specifically bind to each other. An undetermined amount of the same antigen is introduced to a patient’s serum sample. Due to this, the radiolabeled antigen and the unlabeled antigen from the serum compete for binding sites on antibodies. When there is an increase in the amount of unlabeled antigen, there is more binding to its antibody, as a result radiolabeled antigen is replaced and overall, there is a decrease in the ratio of antibody and bound radiolabeled antigen to free radiolabeled antigen. Following the separation of the bound antigens, the radioactivity of the free (unbound) antigen that is still present in the supernatant is determined using a gamma counter. Although this is a reliable and sensitive assay, it is costly, and the radioactive material is toxic. Time-Resolved Fluoroimmunoassay (TR-FIA) is a non-isotopic immunoassay, which uses fluorophore to tag indicator antibodies. The fluorophore generates fluorescence at a distinct wavelength when excited by light, which can be quantified using a time-lapse fluorometer. The target antibody or antigen is marked using the time-resolved fluorescence immunoassay (TR-FIA) technique, which makes use of lanthanide elements with distinctive fluorescence properties and the associated chelates as markers [18, 19]. It frequently makes use of antibody that is adhered to the microplate well’s bottom. The antibody binds to the target molecule on the plate, while the samples are incubated in these wells. After washing the plate to remove any unbound samples, a secondary antibody that is covalently attached to the most commonly used lanthanide chelate, europium, is added, while the secondary antibody that is not attached is washed away. The amount of lanthanide-labeled antibody present is directly correlated with the concentration of the target molecule present in the sample. The auto-fluorescence signal’s decay serves as the time gate for the detection. This indicates that the short-lived auto-fluorescence signal must have completely faded before time-resolved fluorescence detection may begin. Integrated intensity is used to measure the data instead of time decay, and the detected emission signal is integrated across a certain time range. A standard curve can be used to quantify the analyte amount since it is proportional to the time-resolved emission signal. It has the same sensitivity as RIA but requires more expensive equipment [20]. Examples: Varicella Zoster virus and Lyssa virus can be diagnosed by this method (Figure 3).
Figure 3.
Diagrammatic representation of (A) radioimmunoassay and (B) time-resolved fluoroimmunoassay.
2.3.3 Chemiluminescence immunoassay
It is the technique in which the label/conjugate used is the luminescent molecule. Luminescence is the emission of visible or near-visible radiation (300–800 nm), which is generated when an electron transitions from an excited state to a ground state and energy in the atom gets released in the form of light which is measured in relative light unit. Chemiluminescent can be direct using luminophore (acridinium and ruthenium esters) markers or indirect using enzyme markers (alkaline phosphatase with adamantyl 1,2-dioxetane aryl phosphate and horseradish peroxidize with luminal or its derivative as substrate). Antigen or antibodies are directly labeled with chemiluminescent agent and emit light after oxidizing and emission of light is measured. It is more sensitive than ELISA and has a long luminous time, and it takes less time for analysis of sample [21].
2.3.4 Latex particle agglutination
It is the simplest immunoassay available. It is accomplished through the agglutination of tiny latex beads coated with antiviral antibodies with antigen. Collected sample were mixed with latex beads coated with specific antigen or antibody in normal saline. If antigen or antibody of the virus of interest is present, the beads will agglutinate together and the result can be viewed in minutes. However, due to limited sensitivity and specificity, false-negative results are common in samples with insufficient number of virions. Examples: avian influenza virus and Lyssa virus can be diagnosed by this method (Figure 4) [22].
Figure 4.
Diagrammatic representation of (A) positive agglutination test for antibody. (B) Positive agglutination test for antigen.
2.3.5 Immunochromatography
Immunochromatography is used to determine the movement of antigen or complexes of antigen and antibody via support material such as agarose gel, filter paper, or nitrocellulose membrane. A standard format involves tagged antibody to react with an unidentified antigen, and the antigen-antibody complex migrates through the solid support via capillary action, allowing the complex to be detected if antigens are present. Positive control and negative control are used to confirm the validity of individual tests [22]. Examples: Adenovirus [23], Rotavirus, and Norovirus can be diagnosed using this method (Figure 5) [24].
Figure 5.
Diagrammatic representation of immunochromatography. (A) Control test. (B) Positive immunochromatographic test. (C) Negative immunochromatographic test.
2.3.6 Viral cytopathology (cytology/histology)
Rapid diagnosis began with the investigation of viral cytopathology. Herpes virus infections diagnosed by using Tzank preparation are an example of this type of testing. The skin vesicle base is scraped and transferred to a microscope slide. After the slide is dried naturally, it is stained with Wright, Giemsa, or Papanicolaou stain. A normal microscope is used to view the slides. For viruses such as cytomegalovirus (CMV), Varicella zoster virus (VZV), Human Papillomavirus (HPV), BK virus, and parovirus B19, and particular cytological alterations can be validated by staining for specific antigens, antibodies, or nucleic acid probes. It includes immunohistochemistry and in situ hybridization [6].
2.3.6.1 Detection of antigen in tissues (immunohistochemistry)
Antigens from viruses can be detected in sections of frozen tissue or exfoliated cells when tissue or cells are fixed on a glass slide, which serves as a solid support. When activated by shorter-wavelength lights, an antigen and antibody combination that has been tagged by a fluorochrome produces light of a specified long wavelength. In a normal microscope, this light can be seen as fluorescence after other wavelengths have been filtered. However, the sensitivity of this approach is insufficient for detecting fluorescent antibody complexes with soluble antigens or virions. The approach is classified into two distinct subtypes. They are as follows: direct immunofluorescence and indirect immunofluorescence [2, 6, 22]. Examples- Human herpesviruses, Epstein-Barr virus, and cytomegalovirus can be diagnosed by this method (Figure 6) [25].
Figure 6.
Diagrammatic representation of immunohistochemistry. (A) Indirect immunofluorescence. (B) Direct fluorescence.
2.3.7 Complement fixation
In the presence of RBC, the antigen-antibody complex binds complement, rendering it critical for the lysis of RBC. It consists of two stages. The first stage involves mixing guinea pig serum, which includes the complement, with a specific antigen and test serum. The second stage of the response cannot occur if particular antibodies are present because the complement joins with the antigen-antibody complex. The second stage involves adding erythrocytes from sheep that were previously exposed to rabbit serum anti-sheep erythrocytes (hemolytic system). No hemolysis will take place if the complement has been fixed in the first step (positive reaction). On the other hand, the complement, which does not bind, can interact with the hemolytic system and lyse sheep erythrocytes if the blood test does not include appropriate antibodies (a negative reaction) [26]. Examples: Hepatitis virus and Lyssa virus can be diagnosed by this method (Figure 7) [27].
Figure 7.
Diagrammatic representation of (A). Complement fixation (B). No complement fixation.
2.3.8 Immunoperoxidase staining
Similar to IF, immune peroxidase (IP) methods use a conjugate that is an enzyme, like horseradish peroxidase. While an anti-animal species antibody is labeled in the indirect technique, the virus-specific antibody is enzyme-tagged in the direct method. Addition of substrate, such as aminoethyl carbazole or di-amino benzidine, and oxidizing in the presence of hydrogen peroxide result in a reddish-brown color, and the virus-infected cells bound to enzyme conjugate can be observed. IP staining has a number of advantages over IF, although it requires an additional step (the addition of the substrate). With the naked eye or a light microscope, the reaction can be observed. The reagents are more stable, the slides are more permanent, and there are fewer nonspecific reactions [28]. Although endogenous peroxidase, which is found in a variety of tissues, most notably leukocytes, might give a false positive, this issue can be avoided with good procedures and enough controls [22]. Examples: Baculovirus can be diagnosed by this method (Figure 8) [29].
Figure 8.
Diagrammatic representation of immunoperoxidase staining.
2.3.9 Hemagglutination inhibition assay (HIA)
Certain viruses carry an antigen called hemagglutinin on their surface that attaches and agglutinates RBCs. This process is referred to as hemagglutination (HA). The ability of viruses to agglutinate RBCs was used to develop the HI technique. On a microtiter plate, serial dilutions of serum samples are made for the HI test. Following that, a predetermined number of viral hemagglutinins and suitable RBCs were added. In the absence of hemagglutinins, favorable reactions are seen. This is determined by microtiter plate tilting, which allows for the streaming of free RBCs (Figure 9) [2].
Figure 9.
Diagrammatic representation of Hemagglutination inhibition assay. (A). Control assay. (B). Hemagglutination assay. (C). Hemagglutination inhibition assay.
2.3.10 Western blotting analysis
This technique, alternatively called immunoblotting, is used for detecting antiviral antibodies or the viral protein by denaturing and separating entire viral protein on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Then, viral proteins are electro-transferred to nitrocellulose membranes. After that, the membrane is treated with antibodies linked to enzymes specific for the viral proteins. When enzyme-labeled antibodies bind to viral proteins, chromogenic substrate is added, which results in colorful bands at the locations of the viral antigens [2, 22]. Examples: Lyme disease virus, human immunodeficiency virus (human immunodeficiency), and Hepatitis B virus can be diagnosed by this method (Figure 10).
Figure 10.
Diagrammatic representation of Western blotting assay.
2.3.11 Neutralization assays
Neutralization assays for viruses are much specialized assays that neutralize antibodies. They are also employed to verify the results of other types of serological assays, like ELISA, that have been shown to cross-react with viruses belonging to the same family, for example, the Flaviviruses (Dengue virus, West Nile virus, and Zika virus). Antigen is combined with antibody dilutions, and the prevention of cytopathic effects (CPE) is observed via inoculation on a monolayer of tissue. The inhibitory impact can be determined either by observing the CPE or by overlaying agar that allows for plaque formation in plaque reduction neutralization test (PRNT) [30].
2.3.11.1 Plaque reduction neutralization test (PRNT)
The plaque reduction neutralization test is used to determine the titer of antiviral antibodies. A viral suspension is combined with a diluted serum sample or antibody testing solution. For the interaction of antibodies with the virus, the sample is incubated. To stop the virus from spreading carelessly, carboxymethyl cellulose or agar is applied to the surface of the cell layer. It is possible to estimate the concentration of plaque-producing units by counting the number of plaques (regions of infected cells) that appear after a few days. Depending on the virus, fluorescently labeled antibodies or certain dyes reacting with infected cells are used to measure the plaque-forming units. For lowering the number of plaques by 50%, the amount of serum needed when compared to the serum-free virus provides a measurement of the antibody’s level or potency. The PRNT50 value is used to refer to this measurement. Example: Dengue virus can be diagnosed by this method (Figure 11) [30].
Figure 11.
Diagrammatic representation of plaque reduction neutralization test.
2.3.12 miRNA-based Immunodetection
miRNAs, a class of short conserved non-coding RNAs comprising 19–24 nucleotides, play a crucial role in post-transcriptional regulation. It has been recently found that for various malignancies, exogenous miRNA is potential diagnostic marker, including Hepatocellular Carcinoma (HCC), as part of immunotechniques. In HCC, Alpha-fetoprotein, exosomes, and serum microRNAs have shown promise as diagnostic markers. Another noteworthy study introduced a synthetic miRNA-based approach to produce neutralizing antibodies, which are directly expressed in the lungs through aerosol administration. This method aims to combat Respiratory Syncytial Virus (RSV) and influenza infections in humans. Recent research highlights the evolutionary conservation of endogenous miRNAs, and their presence in body fluids indicates their role in regulating pathogenesis, immune responses, and viral infections. These circulating miRNAs have proven valuable for early diagnosis of viral infections, with diagnostic techniques relying on biomarkers. Numerous circulating microRNA biomarkers have been identified for diagnostic purposes, such as miR29b and miR125 for hepatitis infection, miR155 and miR1260 for influenza, and miR12, miRVP3p, and miR184 for arboviruses. These discoveries have significantly enhanced the early detection and management of viral infections [1, 31].
2.3.13 Metagenomics-based immunodetection
Successful testing of the mNGS pan-pathogen detection assay has shown promise in patients with acute viral infections. To efficiently analyze Next-Generation Sequencing (NGS) data, an innovative bioinformatics pipeline called SURPI+ was developed. This pipeline enables the automatic generation of pathogen summaries and visualization of results through a graphical user interface. In a recent breakthrough, metagenomic next-generation sequencing was applied in diagnosing SLEV meningoencephalitis. Notably, studies reveal that metagenomic viral sequencing has the potential to identify viruses beyond influenza. Respiratory sample sequencing utilizing Oxford Nanopore Technology has been explored, offering significant potential for diagnosing various clinical respiratory viruses when compared to current standard diagnostic approaches. The nanopore platform holds great promise for enhancing virus detection in clinical settings [1, 32].
In recent times, the development of therapeutic and aptamer-based diagnostic platform technologies has shown promise in diagnosing viral infections. Oligonucleotide aptamers, in particular, have emerged as prospective alternatives to monoclonal antibodies for detection. They possess the unique ability to target various proteins within infected cells or viral particles, making them innovative and versatile diagnostic agents. In the future, these aptamer molecules hold the potential to serve as viable alternatives to traditional monoclonal antibodies in diagnostic applications [1, 33].
Microfluidics has enabled significant advancements in the study of viral fusion kinetics, viral infectivity, and the screening of neutralizing molecules for viral responses. Additionally, microfluidic platforms hold great promise as clinical tools for diagnostics and drug screening. The field of microfluidics emphasizes precise control and manipulation of fluids within microscopic channels, supporting various biological research methods, including flow cytometry, DNA analysis, enzyme tests, and immunoassays. Recent research has explored the use of integrated microfluidic systems for diagnostic purposes. For instance, aptamers targeting Influenza A viruses (IAV H1N1) were utilized to detect the virus. The complexity and variability of viral infections, heavily influenced by the chemical and physical environment, make microfluidics an ideal platform for conducting studies on infection biology. This technology offers unique insights and opportunities to advance our understanding of viral infections and develop improved diagnostic and therapeutic approaches (Figure 12) [1].
Figure 12.
Diagrammatic representation of (A) IgG or IgM antibodies, which are detected on microfluidic chip using fluorescence detection. (B) Antigen, which is detected on microfluidic chip using fluorescence detection.
2.3.16 Nanoparticles-based immunodetection
The emerging new class of nanoscale materials, possessing diverse and remarkable features, has sparked a wave of innovation in biological and diagnostic applications. Nanoparticle-based lateral flow immunoassays (LFIAs) are used as a point-of-care diagnostic tool for detecting disorders and infectious viral agents. LIFA is a paper-based point-of-care strip biosensor used to identify specific analytes in virus-infected samples. Detection strategies based on gold nanoparticles (AuNP) have demonstrated effectiveness for various viruses, with special emphasis on utilizing bio-AuNP hybrid structures and employing recently developed assay techniques and formats. Notable examples of viruses, where LFIA and AuNP-based strategies have been applied, include Ebola virus, Dengue virus, Human cytomegalovirus, West Nile virus, Human Papillomavirus, and more. These innovative approaches hold significant promise for rapid and accurate diagnosis in critical healthcare scenarios (Figure 13) [1, 34].
Figure 13.
Diagrammatic representation of nanoparticles-based immunodetection.
2.3.17 Immunosensor-based immunodetection
Immunodetection can be accomplished using an immunosensor. A selective and sensitive electrochemical immunosensor for early-stage diagnostics can be constructed by integrating an immune sensing chip with a miniaturized potentiostat and connecting it to a smartphone. Researchers designed a low-cost, portable graphene-enabled biosensor with an immobilized monoclonal antibody that is highly selective. Field Effect Biosensing (FEB), which depends on covalently attaching monoclonal antibodies to graphene, allows for the real-time quantitative detection of antigens. An electrochemiluminescence (ECL) immunosensor of the sandwich type was developed for the ultrasensitive detection of surface antigens [1]. Examples: Influenza virus can be diagnosed by this method (Figure 14) [35].
Figure 14.
Diagrammatic representation of immunosensor-based immunodetection.
2.3.18 Surface-enhanced Raman spectroscopy (SERS)
Surface-enhanced Raman spectroscopy (SERS) is becoming a vital tool in bioanalytical applications, such as the identification and categorization of pathogenic organisms because it allows for the quick detection of analytes with chemical specificity inherent to vibrational spectroscopy. Driskell et al. [36] used SERS to detect strains of rotavirus by determining their molecular signature. PLS-DA, a chemometric data analysis tool, made it easier to classify viruses on the basis of differences in spectrum. Its application in determining complexities, physical characteristics of molecules is more impressive and highly favorable in comparison of using tagged reporters in an indirect manner with fluorochrome or radioactive materials. The disadvantage is it is labor expensive and time-consuming, depending on the operator’s abilities, so as a result, automation is required. Examples: Influenza A virus subtypes and SARS-COV-2 can be diagnosed by this method (Figure 15).
Figure 15.
Diagrammatic representation of surface-enhanced Raman spectroscopy.
Nucleic acid-based molecular diagnostic methods have brought about a revolutionary change in diagnostic virology. These techniques offer speed, high sensitivity, and specificity, allowing for the diagnosis of nearly all viruses known to cause human illnesses by identifying specific nucleic acid sequences. They enable rapid detection and can simultaneously identify numerous viruses, including those that are challenging to culture, slow-growing, or possess antigenic variants. The extensive adoption of this technology can be attributed to the combination with other improved techniques. Furthermore, continuous advancements in sequencing technologies for nucleic acids, along with the available sequence data in databases, have greatly facilitated the analysis of collected data. This progress has opened up new possibilities in diagnosing viral infections more accurately and efficiently [22].
2.4.1 Polymerase chain reaction
It is the more sensitive approach for detecting the presence of undetectable genome (DNA or RNA) of viruses. It is a procedure that can be used in place of direct hybridization. It is used for the amplification of a short fragment of viral RNA or DNA found in the clinical specimen up to a millionfold, enabling the detection of infectious agents in minute amounts present in a single sample. It can also identify viral RNA by including a pre-processing step that converts RNA to DNA using reverse transcriptase [2, 6, 22].
2.4.2 Reverse transcriptase-PCR (RT-PCR)
It was designed with the intent of amplifying RNA targets. This technology converts viral ribonucleic acid (RNA) to complementary DNA (cDNA) and then amplifies it using a standard polymerase chain reaction (PCR). It has a specificity of 99.9 to 100% and a sensitivity of 73 to 100% for detection of viral infections [2].
2.4.3 Quantitative real-time PCR (qPCR)
Amplification and detection of nucleic acids of the virus are carried out concurrently in this technique. The amplification product is detected based on the quantity of light emitted by the specimen. A thermal cycler is used to measure the specimen’s fluorescence emission. The computer, coupled with thermal cycler via appropriate software, records the information and generates an amplification graphic for each reaction cycle [2, 37].
RT-qPCR, short form for Real-Time Quantitative Reverse Transcription PCR, is a powerful molecular technique that allows for accurate detection and measurement of the amplified product during each cycle of the PCR process. This method became possible with the development of an oligonucleotide probe designed to hybridize specifically with the target sequence. The key to RT-qPCR lies in the use of a target-specific probe, which, during the PCR process, undergoes cleavage due to the 5′ nuclease activity of Taq polymerase. This cleavage occurs only when the probe binds specifically to the targeted sequence, enabling precise and real-time monitoring of the amplification progress. To initiate this process, reverse transcriptase is employed to convert messenger RNA (mRNA) or total RNA into complementary DNA (cDNA). This conversion step is crucial because it allows RNA, which is the intermediate in gene expression, to be converted into DNA, which is more stable and amenable to PCR amplification. Once cDNA is obtained, the qPCR process is carried out using it as the template. During qPCR, the number of copies of the target DNA sequence is exponentially amplified with each cycle, and the fluorescence emitted by the cleaved probe is continuously measured. This fluorescence data is then used to determine the quantity of targeted DNA present in the initial sample, allowing for accurate quantification (Figure 16) [37].
Figure 16.
Diagrammatic representation of two-step RT-qPCR.
2.4.5 Restriction fragment length polymorphisms (RFLPs)
DNA viruses do not have segmented genomes. However, using the appropriate restriction enzymes, it is possible to segment the isolated viral DNA into specific segments for electrophoresis-based RFLP analysis of the various viral strains and variations. Examples: Influenza A virus can be diagnosed by this method (Figure 17) [38].
Figure 17.
Diagrammatic representation of restriction fragment length polymorphism analysis of viral DNA.
The nucleic acid sequence-based amplification (NASBA) process consists of two stages: denaturation followed by temperature labile polymerase-dependent isothermal amplification. Fluorochrome-labeled probes are introduced for real-time observation. Researchers developed multiplex RT-NASBA (Real-Time Nucleic Acid Sequence-Based Amplification) to simultaneously detect Norovirus genogroup II, Astrovirus, and Rotavirus A. Compared to RT-PCR, RT-NASBA offers several advantages. Since it is performed under isothermal conditions, there is no need for cooling and heating, and the use of reverse transcriptase as a separate step is not required. As a result, amplification is considerably faster, and both the sensitivity and specificity of RT-NASBA are higher than RT-PCR. In the working principle of RT-NASBA, the process begins with reverse transcriptase using antisense primer P1, carrying a 5′-overhang T7 polymerase promoter sequence, to produce cDNA. RNase H then hydrolyzes the RNA in the RNA: cDNA hybrid, resulting in single-strand cDNA. In the next step, with the help of reverse transcriptase, the sense primer P2 anneals to the cDNA, generating double-stranded DNA (dsDNA). Binding of T7 RNA polymerase to the promoter site of the dsDNA ends the non-cyclic phase and produces copies of antisense-RNA of the target RNA. During the cyclic phase, primer P2 binds first to the antisense RNA molecule, forming an RNA-cDNA hybrid through reverse transcriptase. Subsequently, RNase H hydrolyzes the RNA, and primer P1 binds to it, producing dsDNA. The process continues as T7 RNA polymerase generates new RNA copies, which serve as templates for further amplification. Throughout the amplification, a molecular beacon continuously keeps in check about the RNA product accumulation in real time. The advantages of RT-NASBA are direct amplification of RNA, high amplification efficiency with exceptional specificity, real-time detection capabilities, and reduced risk of contamination. This technique can be used to detect viruses such as Rotavirus A, Norovirus genogroup II, and Astrovirus (Figure 18) [39].
Figure 18.
Diagrammatic representation of the working of nucleic acid sequence-based amplification technique.
2.4.7 DNA microarrays
This method is having a great impact in the field of detection of nucleic acid of viruses. In DNA microarray diagnosis, a test sample containing fluorescently tagged viral nucleic acids is utilized. The microchip is composed of a solid support matrix with “printed” areas containing several hundred to several thousand distinct oligonucleotides. They are virus-specific. Fluorescence-based detection is used to detect and quantify the outcomes of hybridization between immobilized probes and fluorescently tagged target sequences [2]. Examples: Parainfluenza 3, Poliovirus 1, Respiratory syncytial virus can be diagnosed by this method (Figure 19) [40].
Figure 19.
Diagrammatic representation of DNA microarray technique.
2.4.8 Next-generation sequencing (NGS)
It is a revolutionary approach that allows for direct sequencing of DNA fragments without the requirement for cloning. Bioinformatic tools are used to analyze the sequencing data. For instance, pyrosequencing Roche 454 identifies the pyrophosphate release after nucleotide is incorporated into a DNA polymerization process. Illumina’s next-generation sequencing technologies detect the release of fluorescence from nucleotides inserted during the DNA polymerization process. By measuring the ionic current of RNA/DNA molecules flowing through the nanopores, the Oxford nanopore (MinIon) technology sequences the target nucleic acid [2, 22]. Examples: Porcine circovirus 3, Sparrow Deltacoronavirus, Porcine pegivirus, Porcine nordavirus, Influenza D virus can be diagnosed by this method (Figure 20).
Figure 20.
Diagrammatic representation of next-generation sequencing technology. (A) Emulsion PCR. (B) Solid-phase amplification. (C) Sequencing and imaging. (D) Data analysis using the software that is available or an integrated workflow such as the GATK pipeline.
2.4.9 CRISPR-Cas system-based determination of viral infections
Rapid nucleic acid detection plays a crucial role in various human health and biotechnological applications, including identification of infectious diseases, pathogens in agriculture, and disease-associated circulating DNA or RNA. In this context, CRISPR-Cas-based methods are gaining attention for diagnosing infectious and non-infectious disorders. These innovative approaches have the potential to outperform traditional PCR-based techniques in numerous applications. The CRISPR-Cas system relies on Cas effector proteins that can cleave DNA or RNA. Guide RNAs direct Cas proteins to the specific target site for nucleic acid cleavage. By modifying the guide RNA, researchers can easily target specific sequences, making it a valuable tool for nucleic acid detection. Three primary CRISPR-Cas methods have been proposed: SHERLOCK, DETECTR, and HOLMES [41].
In the type II CRISPR/Cas system, three phases include CRISPR acquisition, crRNA biogenesis, and interference with invasive DNA. Cas nucleases cleave the RNA or DNA at the identification site and some Cas proteins can also cause collateral cleavage (off-target cutting). This collateral cleavage phenomenon can be utilized for nucleic acid detection. Targeted nucleic acids are initially amplified using an isothermal process, often RPA, to aid Cas-mediated detection. In the CRISPR-Cas system, guide RNA specific to the target DNA location is introduced. When guide RNA binds with target DNA, it leads to target cleavage along with collateral cleavage. The collateral activity of the DNA-recognizing Cas protein, Cas12a, cleaves a fluorophore probe, generating a detectable signal in the test. Detection can be achieved through fluorescence measurement or by using a lateral flow strip for quicker results. Among various CRISPR-Cas techniques, the FELUDA test stands out as a breakthrough technique developed in India [42]. This test utilizes the revolutionary CRISPR technology to detect the SARS-CoV-2 virus. The FELUDA test involves RNA extraction and one-step RT-PCR to convert viral RNA to DNA and amplify it. A mixture containing Cas9 protein, guide RNA, and amplified viral DNA is then created. The Cas9 protein, guided by the RNA, specifically binds to the matching viral DNA, forming a complex. A piece of paper, similar to a home pregnancy test, is used for lateral flow detection, showing a single line for a negative result and a double line for a positive result within 45 minutes. Although the FELUDA test offers rapid results, the initial RNA extraction and RT-PCR steps must be performed in a laboratory, preventing it from being conducted at home (Figure 21) [43].
Figure 21.
Diagrammatic representation of CRISPR Cas technique. Nucleic acid detection in (A) SHERLOCKv2, (B) SHERLOCK, (C) DETECTR, and (D) FELUDA assays using CRISPR-Cas technology.
2.4.10 Mass spectrometry
Mass spectrometry has been successful at significantly improving the diagnosis of many diseases. Present-day mass spectral techniques were first developed by Aston and Dempster in the 1900s. For viral diagnosis at the cellular level, they were applied. A combined assay of multiplex PCR and MALDI-TOF MS was developed to detect multiple viruses simultaneously in a single sample. PCR combined with mass spectrometry is better as compared to standard methods in cases where multiple viruses are present [44]. Example: SARS-COV-2 can be diagnosed by this method (Figure 22) [45].
Figure 22.
Diagrammatic representation of mass spectrometry.
2.4.11 Biosensors
Biosensors allow rapid virus detection in a wide range of samples. The selection of the appropriate membrane forms an important part of biosensor construction. Many studies have employed organic and inorganic materials such as NaN3, nitrocellulose, nylon, and polyether-sulfonate. The detection limit is the primary goal of all biosensors, regardless of whether the technique of detection is the full virus or any protein. Molecular motor biosensor based on F0F1-ATPase, a molecular motor created by using a biotin-streptavidin method to attach the probe to F0F1-ATPase, is one of the biosensors for the detection of viruses. Detection of food-borne enteric viruses can be done in less than an hour by this technology [46]. For the identification of enteric pathogens, a photoluminescence-based immuno-biosensor was built with a graphene oxide array and the detection of Rotavirus was done using the fluorescence resonance energy transfer (FRET) method [47].
It has high sensitivity and relies on specific complementary single-stranded DNA target sequences being hybridized by DNA probes mounted on gold nanoparticles. The main benefit of this method is that its efficiency is unaffected by low ion and molecule concentrations. Its reference database must be updated on a regular basis, and it can be insufficiently sensitive for detection of every mutation. Gold electrode covered by high-affinity binding peptide of Norovirus, which is highly sensitive, was developed by Baek et al. [48]. Enteric viruses can be detected on-site by using an electrochemical biosensor because they are easier to detect and have a high sensitivity. In case of endemic breakout, it is ideal for mass deployment due to its easy usage. Examples: Respiratory Syncytial virus, Influenza virus, and SARS-COV-2 can be diagnosed by this method (Figure 23) [49].
Figure 23.
Diagrammatic representation of working of biosensors.
2.4.12 Aptamers
Aptamers are a stretch of peptides or oligonucleotides that have got a lot of consideration from researchers and scientists all around the world as an alternative to antibodies. They have all of the characteristics of antibodies and are also thermostable and cost-effective, making them ideal for usage in resource-constrained contexts [50]. Aptamers are combined with RT-PCR for successful diagnosis, and utilized as Aptamer-linked immunosorbent assays, cantilever-based aptasensors, and SPR-based similar to ELISA. Due to their very high sensitivity and portability, ease of use, and rapid detection, aptasensors offer the potential for use in point-of-care detection systems [51]. In situ, capture RT-qPCR technique-based aptamers have recently been developed for the identification of human noroviruses from clinical samples [52]. Examples: Human norovirus, Dengue virus, Ebola virus, etc., can be diagnosed by this method (Figure 24) [53].
Figure 24.
Diagrammatic representation of working of Aptamers. (A). Aptamer library is created and attached to target protein. (B). Protein with its specific aptamer is selected and non-specific aptamer is removed by washing. (C). Aptamers bounded with protein are eluted and purified. (D). Characterization of selected aptamer is done. (E). This unbound Aptamer is then amplified for creation of library. (F). Unbound Aptamers are single-stranded DNA, and it is converted into double-stranded DNA after final selection. (G). After conversion, next-generation sequencing is done. (H) Negative selection is done using negative control protein; if unbound aptamers bind to negative protein, then it is discarded.
Surface plasmon resonance (SPR) spectroscopy is a good method to monitor non-covalent chemical interactions in real time and without any interference. It serves as a label-free assay, producing a visible or fluorescent signal without the need for tags, dyes, or specialized reagents like enzyme-substrate complexes. This attribute makes SPR an excellent tool for studying diverse non-covalent interactions, such as those between proteins and DNA, proteins and cells, RNA and DNA, proteins and carbohydrates, receptor-inhibitor complexes, proteins and peptides, and self-assembled monolayers. SPR has found extensive applications in various fields, including clinical immunogenicity studies, drug discovery, and ligand fishing. The principle of SPR involves a light beam passing through a prism and interacting with a metal surface, typically gold or silver. At the interface between these two media, plasmons, or electron charge density waves, are generated, facilitating the real-time monitoring of interactions occurring at the surface. This unique capability of SPR makes it a valuable technique for investigating molecular interactions in diverse research areas [54].
When the metal surface (usually gold or silver) is hit by polarized light at the interface of two mediums with different refractive indices, Surface plasmons, also known as oscillations of free electrons on the metal surface, are excited and detected by SPR techniques. With the Kretschmann-Raether setup, light is focused via a prism onto a metal surface, where the reflection of light is measured. The metal surface absorbs light as the plasmons are set to resonate at a specific incidence angle or resonant angle. This causes a decrease in reflection strength and a dark band to appear in the detector. Most often, proteins are immobilized on the metal surface, using a series of flow cells, and prospective ligands are then introduced over the surface. The resonant angle of the least intensity of light reflected during these biomolecular interactions is identified. The molecules’ binding causes this resonant angle to alter. The SPR sensorgram records these interactions in real time. Its utility has surpassed that of conventional viral detection techniques, particularly in healthcare and medical diagnosis. With the identification of antibodies against various antigens found in the virus, SPR studies were used to identify different Epstein-Barr virus phases. SPR was also used to monitor binding kinetics of dengue antibody with corresponding dengue antigen in real time. Yet, major barriers to reaching the full potential of SPR include the need for sophisticated infrastructure and trained personnel (Figure 25) [55].
Figure 25.
Diagrammatic representation of surface plasmon resonance.
2.5 Other nucleic acid isothermal amplification methods
Other PCR variants do not need high temperatures (95°C) for denaturing the DNA and binding of new primers. These techniques depend on enzymes to displace the strand, including Reverse Transcriptase-PCR (RT-PCR), Nucleic Acid Sequence-Based Amplification (NASBA), Polymerase Chain Reaction (PCR), and Quantitative Real-Time Polymerase Chain Reaction (qPCR) among others. These techniques may have the advantage of being incredibly rapid and requiring no thermal cycling equipment [2].
The loop-mediated isothermal amplification assay (LAMP) method efficiently detects DNA and RNA viruses in human specimens with high sensitivity, specificity, speed, and cost-effectiveness. This approach involves the use of at least six distinct primers (B3, F3, FIP, BIP, LB, LF) and a DNA polymerase with strand-displacement activity. By incorporating reverse transcriptase, RNA targets can also be amplified [2]. LAMP’s targeted amplification relies on multiple primers recognizing 6–8 unique sections of the target DNA, creating a loop structure that facilitates successive rounds of amplification. Viruses detected by LAMP technique include Foot and mouth disease, Norovirus, Human Papillomavirus, Cytomegalovirus, and Human immunodeficiency virus (Figure 26) [56].
Figure 26.
Diagrammatic representation of loop-mediated isothermal amplification assay (LAMP).
2.5.2 TaqMan probe-based insulated isothermal PCR
In this Rayleigh-Bénard convective PCR method, annealing, denaturation, and extension occur in separate zones in the cylindrical tube at a fixed temperature at the bottom. Vessels are insulated using jacket insulators to reduce heat loss. At the bottom of the vessel, DNA denaturation occurs; in the upper zone, annealing of primer occurs; and in the center, extension occurs. This method is simple, inexpensive, rapid, and sensitive to be utilized as a reliable diagnostic tool. Fluorescently tagged probes are included with the primers to allow for real-time monitoring of the amplification. This technique has been used to detect the CPV-2 virus (Canine Parvovirus-2) (Figure 27) [57].
Figure 27.
Diagrammatic representation of Taqman probe-based amplification technique.
Binding proteins and bacterial recombinase enzymes are used in recombinase polymerase amplification, which is usually done at a slightly higher temperature than normal to avoid non-specific amplification. It has revolutionized nucleic acid detection and is a good alternative to PCR. This technique requires only half an hour and no requirement for the template to be thermally denatured. Thermal annealing and denaturation are not used in RPA. Three essential proteins, single-stranded nucleic acid binding recombinase, strand-displacement DNA polymerase, and a single-stranded DNA binding protein are needed for RPA processes [58, 59]. The recombinase binds to a primer that is roughly 30–35 nucleotides long to begin the amplification reaction, after which, in the double-stranded DNA template, the complex searches for the target site. After the identification of the site by complex, it starts a chain exchange reaction right away to create a D-shaped loop. The shifted DNA chain is then bound by a single-stranded binding protein to stop primer dissociation. Recombinase-primer complex causes active hydrolysis of ATP, leading to change in the complex’s structure. The strand-displacement DNA polymerase identifies the 3′ end of primer after separation of recombinase from the nucleoprotein filament. DNA amplification reaction occurs when DNA polymerase inserts the matching nucleotide into the primer’s 3′ end, following the template sequence. With both forward and reverse primers, the amplification response can happen simultaneously in both directions. A fresh template can be created using the synthesized amplicon to accomplish exponential amplification [60]. But still, there are some shortcomings; to prevent smearing caused by the inclusion of additional components, RPA products typically need to be purified before agarose gel electrophoresis. The design and screening of RPA primers and probes cannot be done using specialized software. Therefore, time-consuming and expensive synthesis and screening experiments are needed. Furthermore, the RPA reaction is incompatible with traditional real-time PCR probes (such TaqMan probes), as they are susceptible to give false-positive results. Due to its isothermal amplification characteristics and use of time threshold rather than a cycling threshold, managing real-time amplification with RPA is difficult. As a result, original circumstances, incubation temperature, and mixing procedures all affect the responses [61]. Apart from that due to its high fidelity, portability, affordability, simplicity, sensitivity to inhibitors, and ease of use, this technique is good [62]. Examples: Influenza A virus, Dengue virus, West Nile virus, etc., can be diagnosed by this method (Figure 28).
Figure 28.
Diagrammatic representation of working of recombinase polymerase recombination technique.
2.5.4 Single primer isothermal amplification (SPIA)
In this technique, RNA functions as a primer in a chimeric DNA/RNA sequence, when RNase H degrades the 3′ end of RNA. And to stop the reaction, blockers are used. After sequence amplification, a short-oligonucleotide sequence is used as a blocker to stop further extension. RNase H digests the RNA sequence during amplification and the primers take the vacated position in the following round. SYBR Green II is employed for the identification process when the amplified product binds to single-stranded DNA. Even 1 ng of mRNA can be amplified using this approach. The major drawback is that because RNase H prevents the reaction from being amplified, and it must be confirmed on agarose gel electrophoresis. This method is excellent when it is not possible to design a robust primer [63]. RNase H, a DNA polymerase with high strand-displacement activity, and a single target-specific chimeric primer of ribonucleotides at its 5′ end and deoxyribonucleotides at its 3′ end are all used in SPIA. In the target DNA molecule, chimeric primer hybridizes to a complementary sequence for the amplification process to begin. The target DNA strand is extended by DNA polymerase, beginning with the hybridized primer. When the primer extension begins, RNase H cleaves the 5′RNA part of extension primer (DNA-RNA hybrid), so that the primer binding site left in the target DNA strand can bind to the new chimeric primer. The newest binding primers compete with previously extended primers to control binding to the complementary target DNA sequence. DNA polymerase binding stabilizes the newly attached primer and removes the 5′ end of the previously extended one. When the primer extension restarts replication, 5′ of RNA component of the newly extended primer is cleaved by RNase H, so that the primer binding site is freed for the next replication cycle. Up to 10,000 replications are initiated and repeated using the original transcripts. With this method, amplification of many nucleic acid types is possible [64, 65]. Examples: Bovine Coronavirus can be diagnosed by this method (Figure 29) [66].
Figure 29.
Diagrammatic representation of working of single primer isothermal amplification.
2.5.5 Polymerase spiral reaction (PSR)
The PSR approach was invented lately and was first applied to E.coli BL-21 cells for amplification of blaNIM–1 plasmid. It has rapidity, high specificity, and efficiency under isothermal condition with requirement of one pair of primers and an enzyme. The resemblance of this technique is like a traditional isothermal PCR that is done at a fixed temperature of 61–65°C with a few tweaks to the primer design. The 5′ end of primers has an exogenous area that is 22–25 bases long in both forward and reverse directions. One primer’s exogenous region is oriented in the opposite way as the other’s exogenous region. Using turbidity and/or fluorescent dye (SYBR Green)-based imaging, the amplification of this reaction can be observed in real time and results in the formation of a complex spiral structure. This technique is a cost-effective and convenient alternative for clinical screening, primary quarantine purposes, and on-site diagnosis [67]. Examples: West Nile virus [68] and Porcine circovirus type 3 [69] can be diagnosed by this method (Figure 30).
Figure 30.
Diagrammatic representation of working of polymerase spiral reaction technique.
2.5.6 Quantitative polymerase chain reaction (qPCR) based on primer-probe energy transfer
It utilizes Förster resonance energy transfer (FRET) for simultaneous detection and quantification of viral genome copies. Primer-probe energy transfer (PriProET) concept is employed, enabling the detection of multiple viral variants simultaneously. PriProET offers flexibility in detecting various target nucleic acids, even with changes in the targeted regions and mutations in the viral genome [70]. In this method, the probe sequence is 25–30 nucleotides long, and quencher dye (TAMRA) is used to tag 3′ end, and fluorescent reporter dye (FAM) is used to tag 5′ end [71]. TaqMan probes are designed to anneal within a region of DNA that has been amplified by a particular set of primers. To improve their binding affinity to the target sequence, minor groove binder (MGB) moiety was conjugated with TaqMan probes. The Taq polymerase’s 5′ to 3′ exonuclease activity breaks down the probe, which has annealed with the template as it extends the primer and creates the nascent strand. The fluorophore is released from the probe during degradation, which also breaks the probe’s proximity to the quencher, and quenching effect is eased and fluorescent from fluorophore is released. As a result, DNA template amount present in the PCR and the amount of fluorophore emitted precisely correlate with the fluorescence detected in the quantitative PCR thermal cycler. Examples: Respiratory syndrome virus, Hepatitis E virus, Feline coronavirus [71], snd Porcine coronavirus [72] can be diagnosed by this method (Figure 31).
Figure 31.
Diagrammatic representation of working of real-time polymerase chain reaction (qPCR) based on primer-probe energy transfer.
2.5.7 Hybrid capture assay
In this nucleic acid hybridization assay, the samples containing the target DNA are hybridized with Human Papillomavirus RNA (HPV RNA) probe. The formed RNA-DNA hybrids are captured using a microplate coated with DNA-RNA hybrid antibodies. Detection is achieved through chemiluminescence. To detect the hybrids, specific antibodies for DNA-RNA hybrids are coupled with alkaline phosphate, and they react with the hybrids on the microplate. This leads to the generation of a chemiluminescent signal when a chemiluminescent substrate is applied. The signal is amplified as multiple conjugated antibodies bind to each hybrid. The chemiluminescent substrate is then cleaved by alkaline phosphate, resulting in the emission of light. This emitted light is measured with a luminometer, which enables the presence of target DNA to be determined. Example: Human Papillomavirus can be diagnosed by this method (Figure 32) [73].
Figure 32.
Diagrammatic representation of hybrid capture assay.
2.5.8 Proximity ligation assay (PLA)
Proximity ligation assay is a sensitive, homogeneous, and highly specific immunohistochemical technique that combines the sensitivity of PCR with the specificity of ELISA. It has been demonstrated that this method, which was created by Fredrickson and colleagues in 2002, overcomes the challenges associated with trying to visualize and study individual proteins, protein-protein interaction, and post-translational modifications, such as glycosylation, acetylation, and phosphorylation. PLA technology enables the identification of endogenous protein interactions. The detection of protein proximity is the basis for this approach. PLA employs a single set of primary antibodies. These primary antibodies are directed toward proteins of interest, such as two separate proteins or the two epitopes of fusion proteins, whose proximity we want to investigate. Secondary antibodies attached to short DNA oligonucleotides are used to identify primary antibodies generated in various species. The DNA strands hybridize and participate in the rolling circle synthesis of DNA, if the oligonucleotides are close together (theoretically less than 40 nm). Hybridization of fluorescent-labeled oligonucleotides can also be used to detect these DNA copies. The resulting high fluorescence concentration may be easily viewed and quantified under a microscope. Each dot represents one colocalization [74]. Proximity Ligation Assay can be done using either a direct or indirect method. The Ab-Oligo conjugates are used by direct method and the indirect method uses primary antibodies that have not been modified and are identified by secondary Ab-Oligo conjugates. The method can make use of either polyclonal, monoclonal, or a combination of both antibodies. Example: SARS COV-2 can be diagnosed by this method (Figure 33) [75].
Figure 33.
Diagrammatic representation of proximity ligation assay. (A). Direct proximity ligation assay uses antibody pair with primary conjugation. (B). Indirect proximity ligation assay uses secondary antibody conjugate.
2.5.9 Helicase-dependent amplification (HDA)
The Helicase-dependent amplification is the simplest method for isothermal nucleic acid amplification, it uses a helicase to unwind DNA duplexes isothermally rather than heat, in PCR, mimicking the in vivo process of DNA replication. The original HDA system needs two important proteins, single-stranded DNA binding protein (SSB) and MutL, to maintain ssDNA and prevent the complementary strands from binding again. In a method very similar to the PCR without thermal cycling, at 3′ end and 5′ end of the targeted DNA, two oligonucleotide primers hybridize after DNA has been isothermally unwound and stabilized. When deoxynucleotide triphosphates are added, DNA polymerase extends annealed primers and double-stranded amplified product is produced. The target dsDNA is amplified exponentially as the procedure is repeated. HDA needs only two primers and a few proteins to perform; it has a simpler assay method than other isothermal reactions like LAMP. HDA may be utilized with unprocessed samples without the necessity for the extraction of DNA or RNA. Lateral flow-based detection and fluorescent dyes can be used for the detection of amplified products. Examples: Herpes simplex virus, Human immunodeficiency virus, and Human Papillomavirus can be diagnosed by this method (Figure 34) [76].
Figure 34.
Diagrammatic representation of helicase-dependent amplification.
2.5.10 Rolling circle amplification (RCA)
The replication of circular DNA occurs naturally and rolling circle amplification (RCA) imitates this process. In contrast to procedures like helicase-dependent amplification and recombinase polymerase amplification, which amplify linear DNA, this procedure is designed for use in the amplification of circular DNA sequences. In its most basic form, RCA just needs one primer, which is followed by the strand-displacing polymerase that starts replication. The circular DNA repeat sequences make up the linear product known as the amplicon, which is the result. Although exponential amplification is not possible with this RCA technique, the procedure can be changed to allow for rapid, exponential amplification. These methods include using the two-primer hyper-branched RCA technique and another alternative method called multiple-primed RCA, which uses several primers and padlock probe-based RCA, and when desired target is single-stranded linear DNA, these types of probes are used. Either fluorescence or intercalating dyes can be used to detect the end products in real time. The assay design for RCA is relatively simplistic, requiring at most one primer to amplify circular DNA or RNA and not denaturing the target. It can be used for the sequencing of emerging strains, rapid viral genome amplification, and variation analyses between two variants in addition to viral diagnostics [54]. Examples: Ebola virus, Zika virus, Dengue virus [77], SARS-COV-2, Influenza A and B, Papillomavirus type 16, and Human immunodeficiency virus can be diagnosed by this method (Figure 35) [78].
Figure 35.
Diagrammatic representation of rolling circle amplification.
2.5.11 Signal-mediated amplification of RNA technology (SMART)
The three-way junction formation is the foundation of this technology (3WJ). No thermal cycler or target sequence copying is needed; this technique depends on Signal amplification. It produces a signal that is suitable and extremely target-dependent to find targets in DNA or RNA. The SMART has two components, for extension matching single-stranded oligonucleotide probe with a zone defined on target for hybridization of each probe and the other considerably hybridization of shorter region at adjacent spots to the opposite probe. Annealing of two probes with each other in the presence of a specific target forms 3WJ structure. Extension of short probe by Bst DNA polymerase after 3WJ formation by replication of opposite template probe produces double-stranded T7 RNA polymerase promoter sequence. To enable the synthesis of 3WJ, the generated promoter enables T7 RNA polymerase for synthesizing multiple copies of RNA amplicon in the presence of a particular target. Each RNA amplicon has the potential to self-amplify when it binds to second template oligonucleotide (probe for the amplification), and a double-stranded promoter is created when extended by DNA polymerase. Due to this, there is an increase in transcription; as a result, RNA amplicons increase, which can be detected in a real-time format or by Enzyme-linked Oligosorbent Assay (ELOSA). The target sequence is not itself amplified throughout this process, which is a signal amplification technique (Figure 36) [65].
Figure 36.
Diagrammatic representation of signal-mediated amplification of RNA technology (SMART).
There are many diagnostic methods for the diagnosis and detection of human viruses. These diagnostic methods have changed drastically in past decades, but still, there is a gap which leads to wrong interpretation of results. The main reasons for these misinterpretations are cross reactivity, loss of genomic material, contamination of samples, multiple viruses causing same disease, etc. and symptom shown at later stage. However, the advanced techniques have greatly improved the diagnosis of these human viruses but they are cumbersome with lengthy procedures and need expertise. But before detection, awareness and knowledge regarding diseases caused by human viruses are most important. It will become easier to diagnosis these viruses. With proper knowledge and advanced techniques, we can come up with more new handy techniques for the detection of human viruses (Figure 37).
Figure 37.
Image credit: “Prevention and treatment of viral infections”, by OpenStax college, biology, CC BY 4.0. Modification of original work by Mikael Häggströms.
High sensitivity and specificity; combination of different assays in one method; automation of assay; requirement of small sample assay; less time-consuming; rapid and low detection limit; multiplex reaction; rare drug detection; use of next-generation sequencing; low reaction cost; lower risk of contamination.
All detection technology will advance toward more sensitive and less exhaustive methods of disease detection. A greater emphasis should be placed on molecular and immunological diagnosis to facilitate and expedite diagnosis. Viruses evolve at an alarming rate, making them extremely difficult to diagnose and identify. In the near future, a quicker diagnostic tool may be developed. Clinical virology is undergoing a paradigm shift as a result of new diagnostic tools. Molecular diagnostic techniques are more sensitive and specific. However, technological capabilities alone are insufficient; health promotion programs should emphasize the need for early detection of infectious disease outbreaks and their dissemination. Less developed countries are unable to advertise and cannot afford pricey diagnoses; this issue must be resolved by providing all assistance necessary to halt the spread of disease. It is envisaged that in the near future, new high-quality tests will be affordable enough for low-income countries to afford and implement policies for disease control and prevention.
For virus detection, a combination of molecular and serological methods should be employed. Having a thorough understanding of the disease’s pathophysiology and epidemiology will only aid in determining the best course of action for treatment. Emerging and re-emerging viruses should be identified using new approaches. Viral DNA or RNA is detected using molecular techniques far before an antibody reaction is triggered. Viral infection diagnosis and treatment rely heavily on immunoassay; however, these tests are not generally available. Since only wealthy nations can currently afford good-quality diagnostics, the application of cutting-edge techniques in poor nations and disease-endemic regions is now being held up. It is projected that attempts will continue to be made to provide new, low-cost, high-quality tests, which will significantly strengthen disease control tactics. The majority of emerging nations have built necessary departments and institutes to train their nationals both domestically and overseas. There is hope that efforts will continue to be made to provide new inexpensive high-quality test for low-income nations, which would drastically boost their populations’ disease control tactics (Tables 3–5).
Virus samples are grown on cell lines and tested for their ability to infect, and if the cells exhibit cytopathic effect, the virus is isolated and cultured.
Produces further material for study, highly sensitive.
Time-consuming, expensive, not applicable for non-viable virus.
Adenovirus, Cytomegalovirus, Enterovirus, Influenza, Japanese encephalitis, COVID-19.
Serological method
These methods are used to study antigen-antibody interaction and identify them in samples.
Rapid and sensitive, serotype information can be obtained, high sensitivity, readily available diagnostic kit.
For almost all viruses, antigen/antibody cannot be detected, interpretation can be false, low sensitive as compared to PCR.
Rubella virus, Measles, Hepatitis A, Cytomegalo virus.
Polymerase chain reaction (PCR)
Amplification of small segment of genes to produce billions of copies in the in vitro condition in a short period of time.
Rapid, highly sensitive, applicable to all viruses including non-cultivable, multiplexing.
Contamination of DNA, detection of non-relevant co-infections due to high sensitivity, low specificity.
HIV, Hepatitis B and C, Human Papillomavirus, Coronavirus.
Latex particle Agglutination (LPA)
Formation of visible clump when antibody or antigen binds with antigen or antibody immobilized on latex particles.
Less time-consuming, analytic sensitivity is high, provides consistency, uniformity, and stability, easy to interpret.
False negative reaction, amount of binding influenced by pH, osmolarity, ionic concentration.
Rabies, Avian influenza virus.
Radioimmunoassay (RIA)
Competitive binding reaction is the fundamental tenet of RIA, where the analyte competes with an antigen that has been radiolabeled for binding to a fixed antibody or a receptor’s binding sites.
High specificity and sensitivity, can detect small amount of antigens or antibodies.
Radiation hazardous, high cost of waste disposal requires a specially trained person, requirement of special license.
Mumps, Measles, Influenza A, Hepatitis C.
Western blotting (WB)
Interaction of protein and probe or antigen/antibody for detection of protein.
Can detect very small quantity of sample, uses antigens and antisera as diagnostic tool, high specificity and sensitivity.
Non-intended protein can react, high amount of sample can give wrong result, appearance of bubbles while transferring.
Coronavirus, HIV, Flaviviruses.
Virus neutralization test (VNT)
Certain antitoxins or antibodies can lessen or neutralize a variety of biological effects brought on by various viruses or enzymes and toxins released by them.
Specificity is high and measures neutralizing antibodies.
Time-consuming, safety is low, needs to be operated in BSL 3/4.
Rabies, Coronavirus, HIV.
Hemagglutination Inhibition (HI)
Ability of viruses to cause red blood cells to agglutinate and agglutination is stopped by antibodies.
High specificity, easy and simple to perform, no instrument required for reading result.
Sensitivity is low, hemagglutinins are not present in all viruses, Interpretation often subjective.
Influenza, Measles, Mumps, Rubella.
Immunofluorescence (IF)
Identification of particular target antigens by using fluorescently-labeled antibodies and enables the use of fluorescent dyes to visualize the distribution of the target molecule using a fluorescence microscope.
High specificity and sensitivity, easy to operate, multiple targets can be detected.
Specialized trainee, cross-reactivity, long storage of immunofluorescence sample cannot be done.
Influenza A and B, Coronavirus
Immunodiffusion (ID)
Insoluble visible lines of precipitate in a gel are made by interaction of soluble antigens and antibody.
Cheap, reliable, repeatable, indicate identity, cross reaction, and non-identity between different antigens.
They are relatively insensitive and are best for detecting viral antigens or antibodies in persistent viral illnesses where viral antigens are present all of the time.
It is a real-time enzymatic amplification method that can identify and amplify RNA even when DNA is present.
Reaction occurs isothermally at 41°C, faster amplification kinetics, three enzyme actions can produce more than 109 copies in just 90 minutes, no risk of DNA contamination
Thermolabile enzyme must be used for isothermal reaction, extraneous nucleic acids may contaminate it.
Use of DNA polymerase with strand-displacement activity is the key principle due to which dsDNA does not need to be denatured to allow for primer annealing and subsequent amplicon elongation.
Rapid, sensitive, increase in specificity by using multiple primers, can be quantitative or qualitative.
Requirement of increased operating temperature mainly of 65 °C; requirement of specific software for designing the primer; risk of contamination.
Coronavirus, Zika virus, Dengue virus, Lassa virus, Hepatitis B.
Proximity Ligation Assay (PLA)
Primary antibodies from different species are directed against two proteins of interest, then the secondary antibodies coupled to DNA probes are added, if the two proteins are close to one another, the hybridized DNA will be used for rolling circle amplification.
Fast, ultra-sensitive, highly efficient, no sample purification needed, suitable for a wide range of sample types, specificity of ELISA, and sensitivity of PCR utilized to produce result.
Dependent on quality of antibody used, non-specific ligation of oligonucleotide can produce background signal, time-consuming, covalent conjugation of oligonucleotide to antibody can be difficult.
Foot and mouth disease virus, Influenza (H1N1).
Recombinase polymerase reaction (RPA)
It is an isothermal amplification alternative to PCR. The RPA process allows for the simultaneous detection of RNA and DNA without the need for a cDNA synthesis step by the addition of enzyme reverse transcriptase.
Rapid, this technique can be performed at body or room temperature, designing of primer is simple; DNA denaturation is not required before amplification.
Lower processing temperature reduces the specificity.
It is an isothermal enzymatic process, where a short DNA or RNA primer is amplified to yield a long single-stranded DNA or RNA using a circular DNA template and particular DNA or RNA polymerases.
Availability of different formats (e.g., hyper-branched, linear, padlock probe), requirement of only one primer.
Depending on the amplification method, product yield can be low, can give false positive result, risk of contamination resulting from either release of probes from the hybridized targets or by the removal of non-specific DNA strands from the target.
Basic principle is a transcription of RNA into complementary DNA by reverse transcriptase from total RNA or mRNA, then used as template for qPCR reaction.
Quantitative, it is highly specific when well-designed primers are used, high sensitivity, for high volume testing, it is amenable.
Requirement of trained staff and specialized equipment, long runtime.
A recombinant form of the Cas9 protein with DNA endonuclease activity attaches to a single guide RNA (sgRNA), consisting of a crRNA sequence unique to the DNA target and a tracrRNA sequence interacting with the Cas9 enzyme. For the intended target, the resultant compound will specifically break double-stranded DNA. The error-prone non-homologous end joining DNA repair mechanism will repair the cleavage site, which could result in insertions or deletions that can affect gene function.
Can be employed alone or with other well-known amplification techniques; due to recognition of target by both the primary amplification technique and guide RNA, this technique provides more specificity, rapid.
Generally requires pre-amplification of target, may have off-target effect where cutting of wrong genes may occur due to Cas9 enzyme, risk of contamination.
Capillary action is used to carry out the immunological reaction on the chromatographic paper. Two types of particular antibodies against the antigen are used. One antibody is immobilized on chromatographic paper, while another is infiltrated into a sample pad and tagged with colloidal gold.
Reliable, simple, rapid, cheap.
Not sensitive, cross-reactivity checking by other tests needed.
Adenovirus, Dengue virus, Puumalavirus.
Microarray
The concept of “nucleic acid hybridization” is the foundation of the DNA microarray. During this process, hydrogen bonds are formed between two complementary strands of DNA to create a double-stranded molecule. This makes it easier for researchers to evaluate and compare identical DNA or RNA molecules.
Analysis of multiple genes, high throughput, comparison of gene expression, can categorize disease into subgroups.
Result always not reproducible, expensive, false positive results, require expertization.
Lymphotropic virus, Hepatitis B and C, Herpes simplex virus, Papillomavirus.
Enzyme-linked immunosorbent assay (ELISA)
Specific antibodies recognize the target antigen and measure the quantity and presence of antigens binding, utilizing a substrate that changes color when the enzyme modifies it.
Availability of various formats (e.g., direct, indirect, sandwich, competitive), the large spectrum of commercially available antibodies makes test design reasonably simple, suited for high-volume testing.
Time-consuming, sample matrix can affect the test, different antibodies may have different target specificities, occurrence of false negative results in early infection window.
Zika virus, Ebola virus, Influenza A virus, Human parechovirus, Hepatitis C, Human immunodeficiency virus.
Table 5.
Summary of diagnostic methods.
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Written By
Biswajyoti Borkakoty, Aniruddha Jakharia, Pratibha Singh and Siraj Ahmed Khan
Submitted: 02 August 2023Reviewed: 18 September 2023Published: 06 March 2024
Open access peer-reviewed chapter - ONLINE FIRST
